Humanized monoclonal antibodies against activated protein c and uses thereof

ABSTRACT

Provided are humanized antibodies that selectively bind to and inhibit activated protein C without binding to or inhibiting unactivated protein C. Methods of treatment employing these antibodies are described herein.

PRIORITY CLAIM

This application is a national phase application under 35 U.S.C. §371 ofInternational Application No. PCT/US2013/072137, filed Nov. 27, 2013,which claims benefit of priority to U.S. Provisional Application Ser.No. 61/731,368, filed Nov. 29, 2012. The entire contents of theabove-referenced disclosures are specifically incorporated herein byreference.

SEQUENCE LISTING

Pursuant to 37 C.F.R. 1.821(c), a sequence listing is submitted herewithas an ASCII compliant text file named “BAYRP0002US_ST25.txt”, created onMay 1, 2015 and having a size of ˜25 kilobytes. The content of theaforementioned file is hereby incorporated by reference in its entirety.

BACKGROUND 1. Introduction

Blood coagulation is a process consisting of a complex interaction ofvarious blood components, or factors, which eventually give rise to afibrin clot. Generally, blood components participating in thecoagulation “cascade” are proenzymes or zymogens—enzymatically inactiveproteins that are converted into an active form by action of anactivator. Regulation of blood coagulation is largely accomplishedenzymatically by proteolytic inactivation of the pro-coagulation factorsVa and VIIIa achieved by activated protein C (aPC) (Esmon, 1989).

Protein C is the precursor to aPC, a potent natural anticoagulant.Protein C is activated by thrombin in complex with thrombomodulin (TM).The activation is augmented by endothelial cell protein C receptor(EPCR). TM and EPCR can be down-regulated due to inflammatory mediators,such as tumor necrosis factor, reviewed by Esmon (1999). TM and EPCRhave also been found to be reduced in some forms of septic shock,meningococcemia in particular. Since EPCR and TM are expressed onendothelium, it is not possible to directly determine how well they arefunctioning without removal of blood vessels.

aPC functions as an anticoagulant by proteolytically cleaving anddownregulating pro-coagulant factors. aPC also serves importantfunctions as an anti-apoptosis agent, an anti-inflammatory molecule anda cytoprotectant. Bleeding disorders where homeostatis is dysregulatedthrough a loss of a key factor, such as the absence of Factor VIII inhemophilia, or in trauma patients where the wound process results in atemporary loss of hemostasis, can be treated by the removal of aPC. Suchtreatment, however, could result in unwanted detrimental consequences ofremoving the beneficial functions of aPC in addition to the removal ofthe anti-coagulant activity. Therefore it is desirable to have atherapeutic that selectively targets the anti-coagulant activity of aPCwhile leaving other functions of the molecule intact.

SUMMARY

Thus, there is provided an antibody comprising (a) a heavy light chaincomprising heavy light chain CDRs represented by SEQ ID NOS: 1, 2 and 3;and (b) a light heavy chain comprising light heavy chain CDRsrepresented by SEQ ID NOS: 4, 5 and 6. The antibody maybe a humanizedantibody, and may have the following sequence composition:

TABLE 1 Antibody Sequences FR₁ CDR1 FR₂ CDR2 FR₃ CDR3 FR₄ Light ChainCDR SEQ ID NO: 1 2 3 Heavy Chain CDR SEQ ID NO: 4 5 6 Light ChainFramework Residues* 1-23 39- 61- 102- 53 92 112 Heavy Chain FrameworkResidues** 1-30 36- 69- 108- 49 100 118 *Residues in reference to SEQ IDNO: 26 **Residues in reference to SEQ ID NO: 16

The heavy chain framework regions may be represented by SEQ ID NOS: 7,8, 9 and 10, amino acid residues 1 to 30, 36 to 49, 69 to 100, and 108to 118 of SEQ ID NO: 16 or having 5 or fewer conservative amino acidsubstitutions thereto, and/or the light chain framework regions may berepresented by SEQ ID NOS: 11, 12, 13 and 14 amino residues 1 to 23, 39to 53, 61 to 92 and 102 to 112 of SEQ ID NO: 26, or having 5 or fewerconservative amino acid substitutions thereto. For example residue 14 49of SEQ ID NO: 8 16 may be substituted with Ala, and/or residues 11, 13and 31 of SEQ ID NO: 9 79, 81 and 99 of SEQ ID NO: 16 may be substitutedwith Serine, Valine and Isoleucine, respectively; and/or the heavy chainmay comprise SEQ ID NOS: 16-24 NO: 16, 17, 18, 19, 20, 21 or 22. Alsofor example, residue 4 of SEQ ID NO: 11 26 may be substituted withLeucine; and/or residue 12 72 of SEQ ID NO: 13 26 may be substitutedwith Arginine; and/or the light chain comprises SEQ ID NOS: 26-30 NO:26, 27, 28 or 29. The antibody may be a single-chain antibody or anantibody fragment, such as a Fab′, Fab, F(ab′)₂, a single domainantibody, Fv, or scFv. Also provided is a pharmaceutical compositioncomprising any of the foregoing embodiments dispersed in apharmaceutically acceptable carrier.

The disclosure also provides an expression construct, cell or cell linecomprising a nucleic acid encoding an antibody comprising (a) a heavylight chain comprising heavy light chain CDRs represented by SEQ ID NOS:1, 2 and 3; and (b) a light heavy chain comprising light heavy chainCDRs represented by SEQ ID NOS: 4, 5 and 6. The antibody may be ahumanized antibody. The heavy chain framework regions may be representedby SEQ ID NOS: 7, 8, 9 and 10, amino acid residues 1 to 30, 36 to 49, 69to 100, and 108 to 118 of SEQ ID NO: 16 or having 5 or fewerconservative amino acid substitutions thereto, and/or the light chainframework regions may be represented by SEQ ID NOS: 11, 12, 13 and 14,or having 5 or fewer conservative amino acid substitutions aminoresidues 1 to 23, 39 to 53, 61 to 92 and 102 to 112 of SEQ ID NO: 26.For example residue 14 49 of SEQ ID NO: 8 16 may be substituted withAla, and/or residues 11, 13 and 31 of SEQ ID NO: 9 79, 81 and 99 of SEQID NO: 16 may be substituted with Serine, Valine and Isoleucine,respectively; and/or the heavy chain may comprise SEQ ID NOS: 16-24 NO:16, 17, 18 , 19, 20, 21 or 22. Also for example, residue 4 of SEQ ID NO:11 26 may be substituted with Leucine; and/or residue 12 72 of SEQ IDNO: 13 26 may be substituted with Arginine; and/or the light chaincomprises SEQ ID NOS: 26-30 NO: 26, 27, 28 or 29. The antibody may be asingle-chain antibody or an antibody fragment, such as a Fab′, Fab,F(ab′)₂, a single domain antibody, Fv, or scFv.

Also provided is method of inhibiting activated protein C anticoagulantactivity in a subject, comprising administering an effective amount ofan antibody according to the description above.

Also provided is a method of inhibiting activated protein C amidolyticactivity in a subject comprising administering an effective amount of anantibody according to the description above.

Also provided is a method of treating a subject in need of bloodcoagulation comprising administering an effective amount of an antibodyaccording to the description above.

Also provided is a method of treating a subject suffering from sepsiscomprising administering an effective amount of an antibody according tothe description above. The method may further comprise administration ofactivated protein C.

Also provided is a method of treating a subject suffering fromhemophilia comprising administering an effective amount of an antibodyaccording to the description above.

Also provided is a method of modulating hemostasis in a subject,comprising administering an effective amount of an antibody according tothe description above. The subject may be a trauma patient.

Also provided is a method of modulating thrombosis in a subject,comprising administering an effective amount of an antibody according tothe description above.

Yet another embodiment includes a kit comprising an antibody accordingto the description above. The antibody may be labeled, such as with afluorophore, a radiolabel, a chemiluminescent label, a dye, a quantumdot, a bead or a chromophore. The kit may further comprise a buffer ordiluent, and/or instructions on the use of said antibody. The antibodymay be present in an aqueous suspension, or be lyophilized.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any compound, method, or composition,and vice versa.

Other objects, features and advantages will become apparent from thefollowing detailed description. It should be understood, however, thatthe detailed description and the specific examples, while indicatingspecific embodiments, are given by way of illustration only, sincevarious changes and modifications within the spirit and scope willbecome apparent to those skilled in the art from this detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure. The disclosure may be better understood by reference to oneor more of these drawings in combination with the detailed descriptionof specific embodiments presented herein.

FIG. 1—SEC analysis of 1573 humanized antibodies.

FIG. 2—Immobilization of capture antibody to CM5 chip using aminecoupling method. 9200 RU of anti-mouse FC IgG signal (top figure) and6400 RU of anti-human FC IgG (bottom figure) were generatedrespectively. The running buffer was HBS-EP running buffer: 10 mM HEPES,pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% surfactant P20.

FIG. 3—SPR sensor-grams of binding of human aPC to 1573 antibodies:human aPC was injected over 1573 antibody at concentration of 0, 1.25,2.5, 5, 10, 20 nM, respectively, at 30 μl/min for 180 s of associationphase and 500 s of dissociation phase.

FIG. 4—SPR sensor-grams of binding of cyno aPC to 1573 antibodies: cynoaPC was injected over 1573 antibody at concentration of 0, 5, 10, 20,40, 80 nM respectively at 30 μl/min for 180 s of association phase and420 s of dissociation phase.

FIGS. 5—1573 humanized antibodies binding ELISA of human PC and aPC.

FIGS. 6—1573 humanized antibodies binding ELISA of monkey PC and aPC.

DESCRIPTION

The present disclosure relates to the discovery of monoclonal antibodiesthat selectively bind to activated protein C, but not unactivatedprotein C, and specifically inhibit the anti-coagulation activity ofactivated protein C.

Whenever appropriate, terms used in the singular will also include theplural and vice versa. In the event that any definition set forth belowconflicts with the usage of that word in any other document, includingany document incorporated herein by reference, the definition set forthbelow shall always control for purposes of interpreting thisspecification and its associated claims unless a contrary meaning isclearly intended (for example in the document where the term isoriginally used). The use of “or” means “and/or” unless statedotherwise. The use of “a” herein means “one or more” unless statedotherwise or where the use of “one or more” is clearly inappropriate.The use of “comprise,” “comprises,” “comprising,” “include,” “includes,”and “including” are interchangeable and are not limiting. For example,the term “including” shall mean “including, but not limited to.”

The term “Protein C” or “PC” as used herein refers to any variant,isoform, and/or species homolog of Protein C in its zymogen form that isnaturally expressed by cells and present in plasma and is distinct fromthe activated form of Protein C.

The term “activated Protein C” or “aPC” as used herein refers to anactivated form of Protein C that is characterized by the removal andabsence of a 12 amino acid activation peptide present in Protein C as aresult of a thrombin cleavage site.

As used herein, an “antibody” refers to a whole antibody and any antigenbinding fragment (i.e., “antigen-binding portion”) or single chainthereof. The term includes a full-length immunoglobulin molecule (e.g.,an IgG antibody) that is naturally occurring or formed by normalimmunoglobulin gene fragment recombinatorial processes, or animmunologically active portion of an immunoglobulin molecule, such as anantibody fragment, that retains the specific binding activity.Regardless of structure, an antibody fragment binds with the sameantigen that is recognized by the full-length antibody. For example, ananti-aPC monoclonal antibody fragment binds to an epitope of aPC. Theantigen-binding function of an antibody can be performed by fragments ofa full-length antibody. Examples of binding fragments encompassed withinthe term “antigen-binding portion” of an antibody include (i) a Fabfragment, a monovalent fragment consisting of the V_(L), V_(H), C_(L)and C_(H1) domains; (ii) a F(ab′)₂ fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting of the V_(H) and C_(H1) domains;(iv) a Fv fragment consisting of the V_(L) and V_(H) domains of a singlearm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature341:544-546), which consists of a V_(H) domain; (vi) an isolatedcomplementarity determining region (CDR); (vii) minibodies, diabodies,triabodies, tetrabodies, and kappa bodies (see, e.g., Ill et al.,Protein Eng 1997; 10:949-57); (viii) camel IgG; and (ix) IgNAR.Furthermore, although the two domains of the Fv fragment, V_(L) andV_(H), are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the V_(L) and V_(H) regions pair toform monovalent molecules (known as single chain Fv (scFv); see e.g.,Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc.Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies arealso intended to be encompassed within the term “antigen-bindingportion” of an antibody. These antibody fragments are obtained usingconventional techniques known to those with skill in the art, and thefragments are analyzed for utility in the same manner as are intactantibodies.

Furthermore, it is contemplated that an antigen binding fragment can beencompassed in an antibody mimetic. The term “antibody mimetic” or“mimetic” as used herein is meant a protein that exhibits bindingsimilar to an antibody but is a smaller alternative antibody or anon-antibody protein. Such antibody mimetic can be comprised in ascaffold. The term “scaffold” refers to a polypeptide platform for theengineering of new products with tailored functions and characteristics.

As used herein, the term “anti-aPC antibody” refers to an antibody thatspecifically binds to an epitope of aPC. When bound in vivo to anepitope of aPC, the anti-aPC antibodies disclosed herein augment one ormore aspects of the blood clotting cascade.

As used herein, the terms “inhibits binding” and “blocks binding” (e.g.,referring to inhibition/blocking of binding of aPC substrate to aPC) areused interchangeably and encompass both partial and complete inhibitionor blocking of a protein with its substrate, such as an inhibition orblocking by at least about 10%, about 20%, about 30%, about 40%, about50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%,about 97%, about 98%, about 99%, or about 100%. As used herein, “about”means+/−10% of the numerical value indicated.

In reference to the inhibition and/or blocking of binding of aPCsubstrate to aPC, the terms inhibition and blocking also include anymeasurable decrease in the binding affinity of aPC to a physiologicalsubstrate when in contact with an anti-aPC antibody as compared to aPCnot in contact with an anti-aPC antibody, e.g., the blocking of theinteraction of aPC with its substrates, including Factor Va or withFactor VIIIa, by at least about 10%, about 20%, about 30%, about 40%,about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about96%, about 97%, about 98%, about 99%, or about 100%.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.Accordingly, the term “human monoclonal antibody” refers to antibodiesdisplaying a single binding specificity that have variable and constantregions derived from human germline immunoglobulin sequences. The humanantibodies can include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo).

An “isolated antibody,” as used herein, is intended to refer to anantibody which is substantially free of other biological molecules,including antibodies having different antigenic specificities (e.g., anisolated antibody that binds to aPC is substantially free of antibodiesthat bind antigens other than aPC). In some embodiments, the isolatedantibody is at least about 75%, about 80%, about 90%, about 95%, about97%, about 99%, about 99.9% or about 100% pure by dry weight. In someembodiments, purity can be measured by a method such as columnchromatography, polyacrylamide gel electrophoresis, or HPLC analysis. Anisolated antibody that binds to an epitope, isoform or variant of humanaPC can, however, have cross-reactivity to other related antigens, e.g.,from other species (e.g., aPC species homologs). Moreover, an isolatedantibody can be substantially free of other cellular material and/orchemicals. As used herein, “specific binding” refers to antibody bindingto a predetermined antigen. Typically, an antibody that exhibits“specific binding” binds to an antigen with an affinity of at leastabout 10⁵ M⁻¹ and binds to that antigen with an affinity that is higher,for example at least two-fold greater, than its binding affinity for anirrelevant antigen (e.g., BSA, casein). The phrases “an antibodyrecognizing an antigen” and “an antibody specific for an antigen” areused interchangeably herein with the term “an antibody which bindsspecifically to an antigen.”

As used herein, the term “minimal binding” refers to an antibody thatdoes not bind to and/or exhibits low affinity to a specified antigen.Typically, an antibody having minimal binding to an antigen binds tothat antigen with an affinity that is lower than about 10² M⁻¹ and doesnot bind to a predetermined antigen with higher affinity than it bindsto an irrelevant antigen.

As used herein, the term “high affinity” for an antibody, such as an IgGantibody refers to a binding affinity of at least about 10⁷M⁻¹, in atleast one embodiment at least about 10⁸M⁻¹, in some embodiments at leastabout 10⁹M⁻¹, 10¹⁰M⁻¹, 10¹¹M⁻¹ or greater, e.g., up to 10¹³M⁻¹ orgreater. However, “high affinity” binding can vary for other antibodyisotypes. For example, “high affinity” binding for an IgM isotype refersto a binding affinity of at least about 10⁷M⁻¹. As used herein,“isotype” refers to the antibody class (e.g., IgM or IgG1) that isencoded by heavy chain constant region genes.

“Complementarity-determining region” or “CDR” refers to one of threehypervariable regions within the variable region of the heavy chain orthe variable region of the light chain of an antibody molecule that formthe N-terminal antigen-binding surface that is complementary to thethree-dimensional structure of the bound antigen. Proceeding from theN-terminus of a heavy or light chain, these complementarity-determiningregions are denoted as “CDR1,” “CDR2,” and “CDR3,” respectively [Wu T T,Kabat E A, Bilofsky H, Proc Natl Acad Sci USA. 1975 December;72(12):5107 and Wu T T, Kabat E A, J Exp Med. 1970 Aug. 1; 132(2):211].CDRs are involved in antigen-antibody binding, and the CDR3 comprises aunique region specific for antigen-antibody binding. An antigen-bindingsite, therefore, can include six CDRs, comprising the CDR regions fromeach of a heavy and a light chain V region.

The term “epitope” refers to the area or region of an antigen to whichan antibody specifically binds or interacts, which in some embodimentsindicates where the antigen is in physical contact with the antibody.Conversely, the term “paratope” refers to the area or region of theantibody on which the antigen specifically binds. Epitopes characterizedby competition binding are said to be overlapping if the binding of thecorresponding antibodies are mutually exclusive, i.e., binding of oneantibody excludes simultaneous binding of another antibody. The epitopesare said to be separate (unique) if the antigen is able to accommodatebinding of both corresponding antibodies simultaneously.

The term “competing antibodies,” as used herein, refers to antibodiesthat bind to about, substantially or essentially the same, or even thesame, epitope as an antibody against aPC as described herein. “Competingantibodies” include antibodies with overlapping epitope specificities.Competing antibodies are thus able to effectively compete with anantibody as described herein for binding to aPC. In some embodiments,the competing antibody can bind to the same epitope as the antibodydescribed herein. Alternatively viewed, the competing antibody has thesame epitope specificity as the antibody described herein.

As used herein, “conservative substitutions” refers to modifications ofa polypeptide that involve the substitution of one or more amino acidsfor amino acids having similar biochemical properties that do not resultin loss of a biological or biochemical function of the polypeptide. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), β-branched side chains(e.g., threonine, valine, isoleucine), and aromatic side chains (e.g.,tyrosine, phenylalanine, tryptophan, histidine). Antibodies of thepresent disclosure can have one or more conservative amino acidsubstitutions yet retain antigen binding activity.

For nucleic acids and polypeptides, the term “substantial homology”indicates that two nucleic acids or two polypeptides, or designatedsequences thereof, when optimally aligned and compared, are identical,with appropriate nucleotide or amino acid insertions or deletions, in atleast about 80% of the nucleotides or amino acids, usually at leastabout 85%, in some embodiments about 90%, 91%, 92%, 93%, 94%, or 95%, inat least one embodiment at least about 96%, 97%, 98%, 99%, 99.1%, 99.2%,99.3%, 99.4%, or 99.5% of the nucleotides or amino acids. Alternatively,substantial homology for nucleic acids exists when the segments willhybridize under selective hybridization conditions to the complement ofthe strand. Also included are nucleic acid sequences and polypeptidesequences having substantial homology to the specific nucleic acidsequences and amino acid sequences recited herein.

The percent identity between two sequences is a function of the numberof identical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, such as without limitationthe AlignX™ module of VectorNTI™ (Invitrogen Corp., Carlsbad, Calif.).For AlignX™, the default parameters of multiple alignment are: gapopening penalty: 10; gap extension penalty: 0.05; gap separation penaltyrange: 8; % identity for alignment delay: 40. (further details found atthe world-wide-web atinvitrogen.com/site/us/en/home/LINNEA-Online-Guides/LINNEA-Communities/Vector-NTI-Community/Sequence-analysis-and-data-management-software-for-PCs/AlignX-Module-for-Vector-NTI-Advance.reg.us.html).

Another method for determining the best overall match between a querysequence (a sequence of the present disclosure) and a subject sequence,also referred to as a global sequence alignment, can be determined usingthe CLUSTALW computer program (Thompson et al., Nucleic Acids Res, 1994,2(22): 4673-4680), which is based on the algorithm of Higgins et al.,Computer Applications in the Biosciences (CABIOS), 1992, 8(2): 189-191).In a sequence alignment the query and subject sequences are both DNAsequences. The result of said global sequence alignment is in percentidentity. Parameters that can be used in a CLUSTALW alignment of DNAsequences to calculate percent identity via pairwise alignments are:Matrix=IUB, k-tuple=1, Number of Top Diagonals=5, Gap Penalty=3, GapOpen Penalty=10, Gap Extension Penalty=0.1. For multiple alignments, thefollowing CLUSTALW parameters can be used: Gap Opening Penalty=10, GapExtension Parameter=0.05; Gap Separation Penalty Range=8; % Identity forAlignment Delay=40.

The nucleic acids can be present in whole cells, in a cell lysate, or ina partially purified or substantially pure form. A nucleic acid is“isolated” or “rendered substantially pure” when purified away fromother cellular components with which it is normally associated in thenatural environment. To isolate a nucleic acid, standard techniques suchas the following can be used: alkaline/SDS treatment, CsCl banding,column chromatography, agarose gel electrophoresis and others well knownin the art.

I. Activated Protein C (aPC) and Antibodies

A. Activated Protein C

Protein C is activated by thrombin complexed with thrombomodulin onendothelium. Unlike the few-second transient life of active thrombin invivo, human aPC has about a 20 minute half-life in circulation after itsgeneration (Berg, et al., 2003). Therefore, one can feasibly measure alevel of aPC in plasma to study its regulation under variouspathophysical conditions.

B. Antibodies to aPC

Previously, a murine antibody HAPC1573 was developed which enhancedFL-aPC binding on the endothelial cells. HAPC1573 facilitated aPCinternalization on endothelium through the interaction of Gla domain ofaPC and EPCR on the cells, and this internalization could be blocked byeither EPCR blocking Ab or Gla domain blocking Ab (HPC1575). HAPC1573also dramatically altered the kinetic parameters of aPC toward itschromogenic substrate, Spectrozyme PCa. This profound change of aPCtoward small peptide substrate in the presence of HAPC1573 indicatedthat this mAb recognized an epitope near active site of aPC and theinteraction of Ab and antigen dramatically increased the affinity of APCtoward small peptide substrate but decreased the off rate of productfrom aPC catalytic site. HAPC1573 also almost completely diminished theprolongation effect of aPC in factor Xa initiated one-stage plasmaclotting assay, suggesting that the interaction of HAPC1573 and aPCprevents aPC from cleaving factor Va. Surprisingly, HAPC1573 did notinhibit but actually enhanced aPC cleaving histone H3 and H4.Consistently, HAPC1573 did not inhibit but slightly enhanced aPCcytoprotection activity on endothelium against histone H3 and H4.Finally, their results show that HAPC1573, recognizes aPC, but notProtein C. See U.S. Pat. No. 8,153,766.

Recent studies have shown that anticoagulant activity of aPC isdispensable for its cytoprotective function, but aPC cleavage activitytoward PAR1 might be essential for its anti-apoptotic effect (Mosnier etal., 2004). However, the cytoprotection effect of aPC has been shown notonly in endothelial cells which express EPCR, but also on other cellssuch as neuron and keratinocytes which do not express EPCR on their cellsurfaces (Guo et al., 2004; Berg et al., 2003), indicating othermechanisms than PAR1 mediated aPC signaling might exist.

C. Applications of the Technology

The ability to distinguish between Protein C and aPC demonstrates theutility of antibodies in a convenient ELISA method for measuring aPClevel in plasma in vivo. Typically, it takes less than 4 hours tomeasure a plasma sample containing 1 ng/ml APC with this method comparedto 19 hours or even weeks with enzyme capture assays (Gruber andGriffen, 1992; Liaw et al., 2003).

Also, as discussed above, HAPC1573 altered aPC cleavage activity towarda chromogenic peptide substrate and also blocked aPC anticoagulantactivity in a plasma clotting assay, suggesting this mAb recognizes anepitope near the aPC active site and alters its catalytic activity uponantibody-antigen binding. At the same time, HAPC1573 actually enhancedaPC cleaving extracellular histones, and enhanced APC cytoprotectionactivity on endothelium against histones. This indicates that APCanticoagulant activity for cleaving activated factor V and VIII is notrequired for its cytoprotection activity by cleaving extracellularhistones. Cleaving extracellular histones independent from itsanticoagulant activity might be one of the molecular mechanisms of aPCregulation inflammation and cytoprotection.

Thus, such antibodies against aPC can, for example, be used in treatmentof hemophilia A patients. aPC cleaves both factor VIIIa and factor Vaand thus negatively affects blood clotting. In hemophilia A patients,factor VIII levels are low and the inactivation of factor Va by aPC isprobably a major pathway to regulate hemostasis and thrombosis in thesepatients. Recent clinical reports demonstrated factor V Leiden mutantwhich is resistant to aPC cleavage was beneficial to hemophilia Apatients regarding their bleeding symptom (van't Zant et al., 1997).Blocking aPC anticoagulant activity toward factor Va in vivo with anantibody is an alternative approach for hemophilia A treatments,especially for those patients who have high level factor VIII inhibitorsso that the factor VIII replacement therapy would not be very effective.

In other embodiments, another possible clinical application forantibodies against aPC is in the treatment of trauma patients whereinhomeostasis is disrupted, excessive bleeding is likely, and surgicalintervention is delayed to regain homeostatis. Treatment with antibodiescan selectively restore the pro-coagulant state without eliminating thecytoprotective or anti-inflammatory activities of APC.

Yet another clinical application of antibodies against aPC is incombination with aPC in sepsis treatment. Its bleeding side effect inpatients is due to aPC anticoagulant activity. Because HAPC1573 blockedaPC anticoagulant activity while maintaining, and even enhancing, aPCcytoprotective effect, the mAb-aPC complex can be a better therapeuticthan aPC alone regarding its bleeding side effect.

II. Antibody Structure

Antibodies comprise a large family of glycoproteins with commonstructural features. An antibody is comprised of four polypeptides thatform a three dimensional structure. Typically, an antibody is comprisedof two different polypeptides, the heavy chain and the light chain. Anantibody molecule is comprised of one or more of these units, each unitcomprising two heavy chains and two light chains. An antibody moleculetypically consists of three functional domains: the Fc, Fab, andantigen-binding site.

There are five different types of heavy chain polypeptides designated asα, δ, ε, γ, and μ. There are two different types of light chainpolypeptides designated κ and λ. An antibody typically contains only onetype of heavy chain and only one type of light chain, although any lightchain can associate with any heavy chain.

The carboxyl terminal of each heavy chain polypeptide is known as theconstant (Fc) region. The amino terminal of each heavy and light chainpolypeptide is known as the variable (V) region. Within the variableregions of the chains are hypervariable regions known as complementaritydetermining regions (CDRs). The variable regions of one heavy chain andone light chain associate to form an antigen-binding site. Each heavychain and each light chain includes three CDRs. The six CDRs of anantigen-binding site define the amino acid residues that form the actualbinding site for the antigen. CDR variability accounts for the diversityof antigen recognition.

Antibodies against aPC may be defined by sequences set forth in thefollowing table:

TABLE 1 Antibody Sequences FR₁ CDR1 FR₂ CDR2 FR₃ CDR3 FR₄ Light ChainCDR SEQ ID NO: 1 2 3 Heavy Chain CDR SEQ ID NO: 4 5 6 Light ChainFramework Residues* 1- 39- 61- 102- 23 53 92 112 Heavy Chain FrameworkResidues** 1- 36- 69- 108- 30 49 100 118 *Residues in reference to SEQID NO: 26 **Residues in reference to SEQ ID NO: 16

III. Antibodies Against aPC

A. Antibody Fragments

Thus, in one embodiment, such molecules will comprise fragments (such as(F(ab′), F(ab′)2) that are produced, for example, by the proteolyticcleavage of the mAbs, or single-chain immunoglobulins producible, forexample, via recombinant means. Such antibody derivatives aremonovalent. In one embodiment, such fragments can be combined with oneanother, or with other antibody fragments or receptor ligands to form“chimeric” binding molecules. Significantly, such chimeric molecules cancontain substituents capable of binding to different epitopes of thesame molecule, or they can be capable of binding to an activated proteinC epitope and a “non-activated protein C” epitope.

A single-chain variable fragment (scFv) is another form of antibodyfragment. It comprises a fusion of the variable regions of the heavy andlight chains of immunoglobulins, linked together with a short (usuallyserine, glycine) linker. This chimeric molecule retains the specificityof the original immunoglobulin, despite removal of the constant regionsand the introduction of a linker peptide. These molecules were createdhistorically to facilitate phage display where it is highly convenientto express the antigen binding domain as a single peptide.Alternatively, scFv can be created directly from subcloned heavy andlight chains derived from a hybridoma. Single chain variable fragmentslack the constant Fc region found in complete antibody molecules, andthus, the common binding sites (e.g., protein A/G) used to purifyantibodies. These fragments can often be purified/immobilized usingProtein L since Protein L interacts with the variable region of kappalight chains.

Flexible linkers generally are comprised of helix- and turn-promotingamino acid residues such as alaine, serine and glycine. However, otherresidues can function as well. Tang et al. (1996) used phage display asa means of rapidly selecting tailored linkers for single-chainantibodies (scFvs) from protein linker libraries. A random linkerlibrary was constructed in which the genes for the heavy and light chainvariable domains were linked by a segment encoding an 18-amino acidpolypeptide of variable composition. The scFv repertoire (approx. 5×10⁶different members) was displayed on filamentous phage and subjected toaffinity selection with hapten. The population of selected variantsexhibited significant increases in binding activity but retainedconsiderable sequence diversity. Screening 1054 individual variantssubsequently yielded a catalytically active scFv that was producedefficiently in soluble form. Sequence analysis revealed a conservedproline in the linker two residues after the VH C terminus and anabundance of arginines and prolines at other positions as the onlycommon features of the selected tethers.

The recombinant antibodies against aPC can also involve sequences ormoieties that permit dimerization or multimerization of the receptors.Such sequences include those derived from IgA, which permit formation ofmultimers in conjunction with the J chain. Another multimerizationdomain is the Gal4 dimerization domain. In other embodiments, the chainscan be modified with agents such as biotin/avidin, which permit thecombination of two antibodies.

In a separate embodiment, a single-chain antibody can be created byjoining receptor light and heavy chains using a non-peptide linker orchemical unit. Generally, the light and heavy chains will be produced indistinct cells, purified, and subsequently linked together in anappropriate fashion (i.e., the N-terminus of the heavy chain beingattached to the C-terminus of the light chain via an appropriatechemical bridge).

Cross-linking reagents are used to form molecular bridges that tiefunctional groups of two different molecules, e.g., a stabilizing andcoagulating agent. However, it is contemplated that dimers or multimersof the same analog or heteromeric complexes comprised of differentanalogs can be created. To link two different compounds in a step-wisemanner, heterobifunctional cross-linkers can be used that eliminateunwanted homopolymer formation. An exemplary hetero-bifunctionalcross-linker contains two reactive groups: one reacting with primaryamine group (e.g., N-hydroxy succinimide) and the other reacting with athiol group (e.g., pyridyl disulfide, maleimides, halogens, etc.).Through the primary amine reactive group, the cross-linker can reactwith the lysine residue(s) of one protein (e.g., the selected antibodyor fragment) and through the thiol reactive group, the cross-linker,already tied up to the first protein, reacts with the cysteine residue(free sulfhydryl group) of the other protein (e.g., the selectiveagent).

A cross-linker having reasonable stability in blood can be employed.Numerous types of disulfide-bond containing linkers are known that canbe successfully employed to conjugate targeting andtherapeutic/preventative agents. Linkers that contain a disulfide bondthat is sterically hindered can prove to give greater stability in vivo,preventing release of the targeting peptide prior to reaching the siteof action. These linkers are thus one group of linking agents.

Another cross-linking reagent is SMPT, which is a bifunctionalcross-linker containing a disulfide bond that is “sterically hindered”by an adjacent benzene ring and methyl groups. It is believed thatsteric hindrance of the disulfide bond serves a function of protectingthe bond from attack by thiolate anions such as glutathione which can bepresent in tissues and blood, and thereby help in preventing decouplingof the conjugate prior to the delivery of the attached agent to thetarget site. The SMPT cross-linking reagent, as with many other knowncross-linking reagents, lends the ability to cross-link functionalgroups such as the SH of cysteine or primary amines (e.g., the epsilonamino group of lysine). Another possible type of cross-linker includesthe heterobifunctional photoreactive phenylazides containing a cleavabledisulfide bond such as sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3′-dithiopropionate. The N-hydroxysuccinimidylgroup reacts with primary amino groups and the phenylazide (uponphotolysis) reacts non-selectively with any amino acid residue.

In addition to hindered cross-linkers, non-hindered linkers also can beemployed in accordance herewith. Other useful cross-linkers, notconsidered to contain or generate a protected disulfide, include SATA,SPDP and 2-iminothiolane (Wawrzynczak & Thorpe, 1987). The use of suchcross-linkers is well understood in the art. Another embodiment involvesthe use of flexible linkers. U.S. Pat. No. 4,680,338, describesbifunctional linkers useful for producing conjugates of ligands withamine-containing polymers and/or proteins, especially for formingantibody conjugates with chelators, drugs, enzymes, detectable labelsand the like. U.S. Pat. Nos. 5,141,648 and 5,563,250 disclose cleavableconjugates containing a labile bond that is cleavable under a variety ofmild conditions. This linker is particularly useful in that the agent ofinterest can be bonded directly to the linker, with cleavage resultingin release of the active agent. Particular uses include adding a freeamino or free sulfhydryl group to a protein, such as an antibody, or adrug.

U.S. Pat. No. 5,856,456 provides peptide linkers for use in connectingpolypeptide constituents to make fusion proteins, e.g., single chainantibodies. The linker is up to about 50 amino acids in length, containsat 5 least one occurrence of a charged amino acid (e.g., arginine orlysine) followed by a proline, and is characterized by greater stabilityand reduced aggregation. U.S. Pat. No. 5,880,270 disclosesaminooxy-containing linkers useful in a variety of immunodiagnostic andseparative techniques.

B. Antibody Conjugates

Further provided are antibody conjugates. For both diagnostic andtherapeutic purposes, one can link or covalently bind or complex anagent to an antibody. Such a molecule or moiety can be, but is notlimited to, at least one effector or reporter molecule. A reportermolecule is defined as any moiety which can be detected using an assay.Non-limiting examples of reporter molecules which have been conjugatedto antibodies include enzymes, radiolabels, haptens, fluorescent labels,phosphorescent molecules, chemiluminescent molecules, chromophores,luminescent molecules, photoaffinity molecules, colored particles orligands, such as biotin.

Certain examples of antibody conjugates are those conjugates in whichthe antibody is linked to a detectable label. “Detectable labels” arecompounds and/or elements that can be detected due to their specificfunctional properties, and/or chemical characteristics, the use of whichallows the antibody to which they are attached to be detected, and/orfurther quantified if desired. Another such example is the formation ofa conjugate comprising an antibody linked to a cytotoxic or anticellular agent, and can be termed “immunotoxins.”

Antibody conjugates are used as diagnostic agents. Antibody diagnosticsgenerally fall within two classes, those for use in in vitrodiagnostics, such as in a variety of immunoassays, and/or those for usein vivo diagnostic protocols, generally known as “antibody-directedimaging.”

Many appropriate imaging agents are known in the art, as are methods fortheir attachment to antibodies (see, for e.g., U.S. Pat. Nos. 5,021,236;4,938,948; and 4,472,509, each incorporated herein by reference). Theimaging moieties used can be paramagnetic ions; radioactive isotopes;fluorochromes; NMR-detectable substances; X-ray imaging.

In the case of paramagnetic ions, one might mention by way of exampleions such as chromium (III), manganese (II), iron (III), iron (II),cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III),ytterbium (III), gadolinium (III), vanadium (II), terbium (III),dysprosium (III), holmium (III) and/or erbium (III). Ions useful inother contexts, such as X-ray imaging, include but are not limited tolanthanum (III), gold (III), lead (II), and especially bismuth (III).

In the case of radioactive isotopes for therapeutic and/or diagnosticapplication, one might mention astatine²¹¹, ¹⁴car- bon, ⁵¹chromium,³⁶chlorine, ⁵⁷cobalt, ⁵⁸cobalt, copper⁶⁷, ¹⁵²Eu, gallium⁶⁷, ³hydrogen,iodine ¹²³, iodine¹²⁵, iodine ¹³¹, indium¹¹¹, ⁵⁹iron, ³²phosphorus,rhenium¹⁸⁶, rhenium¹⁸⁸, ⁷⁵selenium, ³⁵sulphur, technicium^(99m) and/oryttrium⁹⁰. ¹²⁵I is often being commonly used in certain embodiments, andtechnicium^(99m) and/or indium¹¹¹ are also often used due to their lowenergy and suitability for long range detection. Radioactively labeledmonoclonal antibodies can be produced according to well-known methods inthe art. For instance, monoclonal antibodies can be iodinated by contactwith sodium and/or potassium iodide and a chemical oxidizing agent suchas sodium hypochlorite, or an enzymatic oxidizing agent, such aslactoperoxidase. Monoclonal antibodies can be labeled withtechnetium^(99m) by ligand exchange process, for example, by reducingpertechnate with stannous solution, chelating the reduced technetiumonto a Sephadex column and applying the antibody to this column.Alternatively, direct labeling techniques can be used, e.g., byincubating pertechnate, a reducing agent such as SNCl₂, a buffersolution such as sodium-potassium phthalate solution, and the antibody.Intermediary functional groups which are often used to bindradioisotopes which exist as metallic ions to antibody arediethylenetriaminepentaacetic acid (DTPA) or ethylene diaminetetraceticacid (EDTA).

Among the fluorescent labels contemplated for use as conjugates includeAlexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL,BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM,Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, RhodamineRed, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or TexasRed.

Another type of antibody conjugates contemplated are those intendedprimarily for use in vitro, where the antibody is linked to a secondarybinding ligand and/or to an enzyme (an enzyme tag) that will generate acolored product upon contact with a chromogenic substrate. Examples ofsuitable enzymes include urease, alkaline phosphatase, (horseradish)hydrogen peroxidase or glucose oxidase. Secondary binding ligands arebiotin and/or avidin and streptavidin compounds. The use of such labelsis well known to those of skill in the art and are described, forexample, in U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;4,277,437; 4,275,149 and 4,366,241; each incorporated herein byreference.

Yet another known method of site-specific attachment of molecules toantibodies comprises the reaction of antibodies with hapten-basedaffinity labels. Essentially, hapten-based affinity labels react withamino acids in the antigen binding site, thereby destroying this siteand blocking specific antigen reaction. However, this can not beadvantageous since it results in loss of antigen binding by the antibodyconjugate.

Molecules containing azido groups can also be used to form covalentbonds to proteins through reactive nitrene intermediates that aregenerated by low intensity ultraviolet light (Potter & Haley, 1983). Inparticular, 2- and 8-azido analogues of purine nucleotides have beenused as site-directed photoprobes to identify nucleotide bindingproteins in crude cell extracts (Owens & Haley, 1987; Atherton et al.,1985). The 2- and 8-azido nucleotides have also been used to mapnucleotide binding domains of purified proteins (Khatoon et al., 1989;King et al., 1989; and Dholakia et al., 1989) and can be used asantibody binding agents.

Several methods are known in the art for the attachment or conjugationof an antibody to its conjugate moiety. Some attachment methods involvethe use of a metal chelate complex employing, for example, an organicchelating agent such as described in U.S. Pat. Nos. 4,472,509 and4,938,948, each incorporated herein by reference). Monoclonal antibodiescan also be reacted with an enzyme in the presence of a coupling agentsuch as glutaraldehyde or periodate. Conjugates with fluorescein markersare prepared in the presence of these coupling agents or by reactionwith an isothiocyanate. In U.S. Pat. No. 4,938,948, imaging of breasttumors is achieved using monoclonal antibodies and the detectableimaging moieties are bound to the antibody using linkers such asmethyl-p-hydroxybenzimidate orN-succinimidyl-3-(4-hydroxyphenyl)propionate.

In other embodiments, derivatization of immunoglobulins by selectivelyintroducing sulfhydryl groups in the Fc region of an immunoglobulin,using reaction conditions that do not alter the antibody combining siteare contemplated. Antibody conjugates produced according to thismethodology are disclosed to exhibit improved longevity, specificity andsensitivity (U.S. Pat. No. 5,196,066, incorporated herein by reference).Site-specific attachment of effector or reporter molecules, wherein thereporter or effector molecule is conjugated to a carbohydrate residue inthe Fc region have also been disclosed in the literature (O'Shannessy etal., 1987). This approach has been reported to produce diagnosticallyand therapeutically promising antibodies which are currently in clinicalevaluation.

In another embodiment, one may choose to modify the immunoglobulins toimprove their stability and half-life in vivo. PEGylation is one suchprocess that involves covalent attachment of polyethylene glycol (PEG)polymer chains to the antibody. PEGylation is routinely achieved byincubation of a reactive derivative of PEG with the target molecule. Thecovalent attachment of PEG can “mask” the antibody from the host'simmune system (reduced immunogenicity and antigenicity), and increasethe hydrodynamic size (size in solution) of the agent which prolongs itscirculatory time by reducing renal clearance. PEGylation can alsoprovide water solubility. Other polymers used to modify antibodiesinclude polyethyleneimine and polylysine, often linked through succinicacid groups.

C. Immunodetection Methods

In still further embodiments, also provided are immunodetection methodsfor binding, purifying, removing, quantifying and/or otherwise generallydetecting biological components using antibodies that reactimmunologically with such components. Some immunodetection methodsinclude enzyme linked immunosorbent assay (ELISA), radioimmunoassay(RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescentassay, bioluminescent assay, and Western blot to mention a few. Thesteps of various useful immunodetection methods have been described inthe scientific literature, such as, e.g., Doolittle and Ben-Zeev (1999);Gulbis and Galand (1993); De Jager et al. (1993); and Nakamura et al.(1987), each incorporated herein by reference.

In general, the immunobinding methods include obtaining a samplecontaining a target of interest, and contacting the sample with a firstantibody that reacts immunologically with the target under conditionseffective to allow the formation of immunocomplexes. The binding of theantibody to the target can then be assessed using a variety of differentformats.

In one format, the antibody can be linked to a solid support, such as inthe form of a column matrix, and the sample suspected of containing thetarget will be applied to the immobilized antibody. The unwantedcomponents will be washed from the column, leaving the targetimmunocomplexed to the immobilized antibody to be eluted.

The immunobinding methods also include methods for detecting andquantifying the amount of an target in a sample and the detection andquantification of any immune complexes formed during the bindingprocess. Here, one would obtain a sample suspected of containing atarget, and contact the sample with an antibody against the target, andthen detect and quantify the amount of immune complexes formed under thespecific conditions.

In terms of antigen detection, the biological sample analyzed can be anysample that is suspected of containing a target, such as, for example, abody fluid like blood, serum, plasma, mucous, urine, saliva, tears orsemen. Alternatively, a tissue can be used. Contacting the chosenbiological sample with the antibody under effective conditions and for aperiod of time sufficient to allow the formation of immune complexes(primary immune complexes) is generally a matter of simply adding theantibody composition to the sample and incubating the mixture for aperiod of time long enough for the antibodies to form immune complexeswith, i.e., to bind to targets that react immunologically withantibodies present. After this time, the sample-antibody composition,such as a tissue section, ELISA plate, dot blot or western blot, willgenerally be washed to remove any non-specifically bound species,allowing only those molecules specifically bound within the primaryimmune complexes to be detected.

In general, the detection of immunocomplex formation is well known inthe art and can be achieved through the application of numerousapproaches. These methods are generally based upon the detection of alabel or marker, such as any of those radioactive, fluorescent,biological and enzymatic tags. U.S. Patents concerning the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated hereinby reference. Of course, one can find additional advantages through theuse of a secondary binding ligand such as a second antibody and/or abiotin/avidin ligand binding arrangement, as is known in the art.

The antibody employed in the detection can itself be linked to adetectable label, wherein one would then simply detect this label,thereby allowing the amount of the primary immune complexes in thecomposition to be determined. Alternatively, the first antibody thatbecomes bound within the primary immune complexes can be detected bymeans of a second binding ligand that has binding affinity for theantibody. In these cases, the second binding ligand can be linked to adetectable label. The second binding ligand is itself often an antibody,which can thus be termed a “secondary” antibody. The primary immunecomplexes are contacted with the labeled, secondary binding ligand, orantibody, under effective conditions and for a period of time sufficientto allow the formation of secondary immune complexes. The secondaryimmune complexes are then generally washed to remove anynon-specifically bound labeled secondary antibodies or ligands, and theremaining label in the secondary immune complexes is then detected.

Further methods include the detection of primary immune complexes by atwo step approach. A second binding ligand, such as an antibody, thathas binding affinity for the antibody is used to form secondary immunecomplexes, as described above. After washing, the secondary immunecomplexes are contacted with a third binding ligand or antibody that hasbinding affinity for the second antibody, again under effectiveconditions and for a period of time sufficient to allow the formation ofimmune complexes (tertiary immune complexes). The third ligand orantibody is linked to a detectable label, allowing detection of thetertiary immune complexes thus formed. This system can provide forsignal amplification if this is desired.

One method of immunodetection designed by Charles Cantor uses twodifferent antibodies. A first step biotinylated, monoclonal orpolyclonal antibody is used to detect the target antigen(s), and asecond step antibody is then used to detect the biotin attached to thecomplexed biotin. In that method the sample to be tested is firstincubated in a solution containing the first step antibody. If thetarget antigen is present, some of the antibody binds to the antigen toform a biotinylated antibody/antigen complex. The antibody/antigencomplex is then amplified by incubation in successive solutions ofstreptavidin (or avidin), biotinylated DNA, and/or complementarybiotinylated DNA, with each step adding additional biotin sites to theantibody/antigen complex. The amplification steps are repeated until asuitable level of amplification is achieved, at which point the sampleis incubated in a solution containing the second step antibody againstbiotin. This second step antibody is labeled, as for example with anenzyme that can be used to detect the presence of the antibody/antigencomplex by histoenzymology using a chromogen substrate. With suitableamplification, a conjugate can be produced which is macroscopicallyvisible.

Another known method of immunodetection takes advantage of theimmuno-PCR (Polymerase Chain Reaction) methodology. The PCR method issimilar to the Cantor method up to the incubation with biotinylated DNA,however, instead of using multiple rounds of streptavidin andbiotinylated DNA incubation, the DNA/biotin/streptavidin/antibodycomplex is washed out with a low pH or high salt buffer that releasesthe antibody. The resulting wash solution is then used to carry out aPCR reaction with suitable primers with appropriate controls. At leastin theory, the enormous amplification capability and specificity of PCRcan be utilized to detect a single antigen molecule.

Another ELISA in which the antigens are immobilized, involves the use ofantibody competition in the detection. In this ELISA, labeled antibodiesagainst an antigen are added to the wells, allowed to bind, and/ordetected by means of their label. The amount of an antigen in an unknownsample is then determined by mixing the sample with the labeledantibodies against the antigen during incubation with coated wells. Thepresence of an antigen in the sample acts to reduce the amount ofantibody against the antigen available for binding to the well and thusreduces the ultimate signal. This is also appropriate for detectingantibodies against an antigen in an unknown sample, where the unlabeledantibodies bind to the antigen-coated wells and also reduces the amountof antigen available to bind the labeled antibodies.

As detailed above, immunoassays, in their most simple and/or directsense, are binding assays. Certain immunoassays are the various types ofenzyme linked immunosorbent assays (ELISAs) and/or radioimmunoassays(RIA) known in the art. Immunohistochemical detection using tissuesections is also particularly useful. However, it will be readilyappreciated that detection is not limited to such techniques, and/orwestern blotting, dot blotting, FACS analyses, and/or the like can alsobe used. Irrespective of the format employed, ELISAs have certainfeatures in common, such as coating, incubating and binding, washing toremove non-specifically bound species, and detecting the bound immunecomplexes. These are described below.

In coating a plate with either antigen or antibody, one will generallyincubate the wells of the plate with a solution of the antigen orantibody, either overnight or for a specified period of hours. The wellsof the plate will then be washed to remove incompletely adsorbedmaterial. Any remaining available surfaces of the wells are then“coated” with a non-specific protein that is antigenically neutral withregard to the test antisera. These include bovine serum albumin (BSA),casein or solutions of milk powder. The coating allows for blocking ofnonspecific adsorption sites on the immobilizing surface and thusreduces the background caused by nonspecific binding of antisera ontothe surface.

In ELISAs, it is probably more customary to use a secondary or tertiarydetection means rather than a direct procedure. Thus, after binding of aprotein or antibody to the well, coating with a non-reactive material toreduce background, and washing to remove unbound material, theimmobilizing surface is contacted with the biological sample to betested under conditions effective to allow immune complex(antigen/antibody) formation. Detection of the immune complex thenrequires a labeled secondary binding ligand or antibody, and a secondarybinding ligand or antibody in conjunction with a labeled tertiaryantibody or a third binding ligand.

“Under conditions effective to allow immune complex (antigen/antibody)formation” means that the conditions can include diluting the antigensand/or antibodies with solutions such as BSA, bovine gamma globulin(BGG) or phosphate buffered saline (PBS)/Tween. These added agents alsotend to assist in the reduction of nonspecific background.

The “suitable” conditions also mean that the incubation is at atemperature or for a period of time sufficient to allow effectivebinding. Incubation steps are typically from about 1 to 2 to 4 hours orso, at temperatures on the order of 25° C. to 27° C., or can beovernight at about 4° C. or so.

D. Purification

In certain embodiments, the antibodies against aPC can be purified. Theterm “purified,” as used herein, is intended to refer to a composition,isolatable from other components, wherein the protein is purified to anydegree relative to its naturally-obtainable state. A purified proteintherefore also refers to a protein, free from the environment in whichit can naturally occur. Where the term “substantially purified” is used,this designation will refer to a composition in which the protein orpeptide forms the major component of the composition, such asconstituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95% or more of the proteins in the composition.

Protein purification techniques are well known to those of skill in theart. These techniques involve, at one level, the crude fractionation ofthe cellular milieu to polypeptide and non-polypeptide fractions. Havingseparated the polypeptide from other proteins, the polypeptide ofinterest can be further purified using chromatographic andelectrophoretic techniques to achieve partial or complete purification(or purification to homogeneity).

Analytical methods particularly suited to the preparation of a purepeptide are ion-exchange chromatography, exclusion chromatography;polyacrylamide gel electrophoresis; isoelectric focusing. Other methodsfor protein purification include, precipitation with ammonium sulfate,PEG, antibodies and the like or by heat denaturation, followed bycentrifugation; gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; and combinations of such and other techniques.

In purifying an antibody against aPC, it can be desirable to express thepolypeptide in a prokaryotic or eukaryotic expression system and extractthe protein using denaturing conditions. The polypeptide can be purifiedfrom other cellular components using an affinity column, which binds toa tagged portion of the polypeptide. As is generally known in the art,it is believed that the order of conducting the various purificationsteps can be changed, or that certain steps can be omitted, and stillresult in a suitable method for the preparation of a substantiallypurified protein or peptide.

Commonly, complete antibodies are fractionated utilizing agents (i.e.,protein A) that bind the Fc portion of the antibody. Alternatively,antigens can be used to simultaneously purify and select appropriateantibodies. Such methods often utilize the selection agent bound to asupport, such as a column, filter or bead. The antibodies is bound to asupport, contaminants removed, and the antibodies released by applyingconditions (salt, heat, etc.).

Various methods for quantifying the degree of purification of theprotein or peptide will be known to those of skill in the art in lightof the present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis. Another method forassessing the purity of a fraction is to calculate the specific activityof the fraction, to compare it to the specific activity of the initialextract, and to thus calculate the degree of purity. The actual unitsused to represent the amount of activity will, of course, be dependentupon the particular assay technique chosen to follow the purificationand whether or not the expressed protein or peptide exhibits adetectable activity.

It is known that the migration of a polypeptide can vary, sometimessignificantly, with different conditions of SDS/PAGE (Capaldi et al.,1977). It will therefore be appreciated that under differingelectrophoresis conditions, the apparent molecular weights of purifiedor partially purified expression products can vary.

IV. Pharmaceutical Compositions and Uses

A. Compositions

Pharmaceutical compositions can comprise an effective amount of one ormore antibodies, therapeutic agents or additional agent dissolved ordispersed in a pharmaceutically acceptable carrier. Aqueous compositionscomprise an effective amount of the antibody, dissolved or dispersed ina pharmaceutically acceptable carrier or aqueous medium. The phrases“pharmaceutically or pharmacologically acceptable” refer to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to an animal, or a human, asappropriate.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated hereinby reference). The use of such media and agents for pharmaceuticalactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active ingredient,its use in the therapeutic compositions is contemplated. Supplementaryactive ingredients can also be incorporated into the compositions. Forhuman administration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologic Standards.

The biological material should be extensively dialyzed to removeundesired small molecular weight molecules and/or lyophilized for moreready formulation into a desired vehicle, where appropriate. The activecompounds will then generally be formulated for parenteraladministration, e.g., formulated for injection via the intravenous,intramuscular, sub-cutaneous, intranasal, or intraperitoneal routes.Typically, such compositions can be prepared as injectables, either asliquid solutions or suspensions; solid forms suitable for using toprepare solutions or suspensions upon the addition of a liquid prior toinjection can also be prepared; and the preparations can also beemulsified.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The antibodies against aPC can be formulated into a composition in afree base, in a neutral or salt form. Pharmaceutically acceptable salts,include the acid addition salts (formed with the free amino groups ofthe protein) and which are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, oxalic, tartaric, mandelic, and the like. Salts formed with thefree carboxyl groups can also be derived from inorganic bases such as,for example, sodium, potassium, ammonium, calcium, or ferric hydroxides,and such organic bases as isopropylamine, trimethylamine, histidine,procaine and the like.

The carrier can also be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, isotonic agents can be included, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate andgelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, the methodsof preparation are vacuum-drying and freeze-drying techniques whichyield a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof. Thepreparation of more, or highly, concentrated solutions for directinjection is also contemplated, where the use of DMSO as solvent isenvisioned to result in extremely rapid penetration, delivering highconcentrations of the active agents to a small area.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms, such as the type of injectable solutions described above,but drug release capsules and the like can also be employed.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, intranasal, and intraperitonealadministration. In this connection, sterile aqueous media which can beemployed will be known to those of skill in the art in light of thepresent disclosure. For example, one dosage could be dissolved in 1 mlof isotonic NaCl solution and either added to 1000 ml of hypodermoclysisfluid or injected at the proposed site of infusion, (see for example,“Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and1570-1580). Some variation in dosage will necessarily occur depending onthe condition of the subject being treated. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual subject.

In addition to the compounds formulated for parenteral administration,such as intravenous or intramuscular injection, other pharmaceuticallyacceptable forms include, e.g., tablets or other solids for oraladministration; liposomal formulations; time release capsules; and anyother form currently used, including cremes.

In certain embodiments, the use of liposomes and/or nanoparticles iscontemplated for the formulation and administration of the antibodiesand/or analogs thereof. The formation and use of liposomes is generallyknown to those of skill in the art, and is also described below.

Nanocapsules can generally entrap compounds in a stable and reproducibleway. To avoid side effects due to intracellular polymeric overloading,such ultrafine particles (sized around 0.1 μm) should be designed usingpolymers able to be degraded in vivo. Biodegradablepolyalkyl-cyanoacrylate nanoparticles that meet these requirements arecontemplated for use, and such particles are easily made.

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs). MLVs generally havediameters of from 25 nm to 4 μm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200-500 Å, containing an aqueous solution in the core.

The following information can also be utilized in generating liposomalformulations. Phospholipids can form a variety of structures other thanliposomes when dispersed in water, depending on the molar ratio of lipidto water. At low ratios the liposome is the recommended structure. Thephysical characteristics of liposomes depend on pH, ionic strength andthe presence of divalent cations. Liposomes can show low permeability toionic and polar substances, but at elevated temperatures undergo a phasetransition which markedly alters their permeability. The phasetransition involves a change from a closely packed, ordered structure,known as the gel state, to a loosely packed, less-ordered structure,known as the fluid state. This occurs at a characteristicphase-transition temperature and results in an increase in permeabilityto ions, sugars and drugs.

Liposomes interact with cells via four different mechanisms: Endocytosisby phagocytic cells of the reticuloendothelial system such asmacrophages and neutrophils; adsorption to the cell surface, either bynonspecific weak hydrophobic or electrostatic forces, or by specificinteractions with cell-surface components; fusion with the plasma cellmembrane by insertion of the lipid bilayer of the liposome into theplasma membrane, with simultaneous release of liposomal contents intothe cytoplasm; and by transfer of liposomal lipids to cellular orsubcellular membranes, or vice versa, without any association of theliposome contents. Varying the liposome formulation can alter whichmechanism is operative, although more than one can operate at the sametime.

The therapeutic agent can comprise different types of carriers dependingon whether it is to be administered in solid, liquid or aerosol form,and whether it needs to be sterile for such routes of administration asinjection. The antibodies against aPC can be administered intravenously,intradermally, intraarterially, intraperitoneally, intralesionally,intracranially, intraarticularly, intraprostaticaly, intrapleurally,intratracheally, intranasally, intravitreally, intravaginally,intrarectally, topically, intramuscularly, intraperitoneally,subcutaneously, subconjunctival, intravesicularlly, mucosally,intrapericardially, intraumbilically, intraocularally, orally,topically, locally, by inhalation (e.g., aerosol inhalation), byinjection, by infusion, by continuous infusion, localized perfusionbathing target cells directly, via a catheter, via a lavage, in cremes,in lipid compositions (e.g., liposomes), or by other methods or anycombination of the foregoing as would be known to one of ordinary skillin the art (see, for example, Remington's Pharmaceutical Sciences, 18thEd. Mack Printing Company, 1990, incorporated herein by reference).

The actual dosage amount of a composition administered to an animalpatient can be determined by physical and physiological factors such asbody weight, severity of condition, the type of disease being treated,previous or concurrent therapeutic interventions, idiopathy of thepatient and the route of administration. The practitioner responsiblefor administration will, in any event, determine the concentration ofactive ingredient(s) in a composition and appropriate dose(s) for theindividual subject.

In certain embodiments, pharmaceutical compositions can comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, an active compound can comprise between about 2% to about75% of the weight of the unit, or between about 25% to about 60%, forexample, and any range derivable therein. In other non-limitingexamples, a dose can also comprise from about 1 microgram/kg/bodyweight, about 5 microgram/kg/body weight, about 10 microgram/kg/bodyweight, about 50 microgram/kg/body weight, about 100 microgram/kg/bodyweight, about 200 microgram/kg/body weight, about 350 microgram/kg/bodyweight, about 500 microgram/kg/body weight, about 1 milligram/kg/bodyweight, about 5 milligram/kg/body weight, about 10 milligram/kg/bodyweight, about 50 milligram/kg/body weight, about 100 milligram/kg/bodyweight, about 200 milligram/kg/body weight, about 350 milligram/kg/bodyweight, about 500 milligram/kg/body weight, to about 1000 mg/kg/bodyweight or more per administration, and any range derivable therein. Innon-limiting examples of a derivable range from the numbers listedherein, a range of about 5 mg/kg/body weight to about 100 mg/kg/bodyweight, about 5 microgram/kg/body weight to about 500 milligram/kg/bodyweight, etc., can be administered, based on the numbers described above.

In any case, the composition can comprise various antioxidants to retardoxidation of one or more component.

In embodiments where the composition is in a liquid form, a carrier canbe a solvent or dispersion medium comprising but not limited to, water,ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethyleneglycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes)and combinations thereof. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin; by the maintenanceof the required particle size by dispersion in carriers such as, forexample liquid polyol or lipids; by the use of surfactants such as, forexample hydroxypropylcellulose; or combinations thereof. In many cases,isotonic agents can be included, such as, for example, sugars, sodiumchloride or combinations thereof.

In other embodiments, one can use eye drops, nasal solutions or sprays,aerosols or inhalants. Such compositions are generally designed to becompatible with the target tissue type. In a non-limiting example, nasalsolutions are usually aqueous solutions designed to be administered tothe nasal passages in drops or sprays. Nasal solutions are prepared sothat they are similar in many respects to nasal secretions, so thatnormal ciliary action is maintained. Thus, in some embodiments theaqueous nasal solutions usually are isotonic or slightly buffered tomaintain a pH of about 5.5 to about 6.5. In addition, antimicrobialpreservatives, similar to those used in ophthalmic preparations, drugs,or appropriate drug stabilizers, if required, can be included in theformulation. For example, various commercial nasal preparations areknown and include drugs such as antibiotics or antihistamines.

In certain embodiments the antibodies are prepared for administration bysuch routes as oral ingestion. In these embodiments, the solidcomposition can comprise, for example, solutions, suspensions,emulsions, tablets, pills, capsules (e.g., hard or soft shelled gelatincapsules), sustained release formulations, buccal compositions, troches,elixirs, suspensions, syrups, wafers, or combinations thereof. Oralcompositions can be incorporated directly with the food of the diet.Carriers for oral administration comprise inert diluents, assimilableedible carriers or combinations thereof. In other embodiments, the oralcomposition can be prepared as a syrup or elixir. A syrup or elixir, cancomprise, for example, at least one active agent, a sweetening agent, apreservative, a flavoring agent, a dye, a preservative, or combinationsthereof.

In certain embodiments an oral composition can comprise one or morebinders, excipients, disintegration agents, lubricants, flavoringagents, and combinations thereof. In certain embodiments, a compositioncan comprise one or more of the following: a binder, such as, forexample, gum tragacanth, acacia, cornstarch, gelatin or combinationsthereof; an excipient, such as, for example, dicalcium phosphate,mannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate or combinations thereof; a disintegratingagent, such as, for example, corn starch, potato starch, alginic acid orcombinations thereof; a lubricant, such as, for example, magnesiumstearate; a sweetening agent, such as, for example, sucrose, lactose,saccharin or combinations thereof; a flavoring agent, such as, forexample peppermint, oil of wintergreen, cherry flavoring, orangeflavoring, etc.; or combinations of the foregoing. When the dosage unitform is a capsule, it can contain, in addition to materials of the abovetype, carriers such as a liquid carrier. Various other materials can bepresent as coatings or to otherwise modify the physical form of thedosage unit. For instance, tablets, pills, or capsules can be coatedwith shellac, sugar or both.

Additional formulations which are suitable for other modes ofadministration include suppositories. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum, vagina or urethra. After insertion, suppositoriessoften, melt or dissolve in the cavity fluids. In general, forsuppositories, traditional carriers can include, for example,polyalkylene glycols, triglycerides or combinations thereof. In certainembodiments, suppositories can be formed from mixtures containing, forexample, the active ingredient in the range of about 0.5% to about 10%,and about 1% to about 2%.

The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein.

In particular embodiments, prolonged absorption of an injectablecomposition can be brought about by the use in the compositions ofagents delaying absorption, such as, for example, aluminum monostearate,gelatin or combinations thereof.

B. Pharmaceutical Uses

The monoclonal antibody can be used for therapeutic purposes fortreating genetic and acquired deficiencies or defects in coagulation.For example, the monoclonal antibodies in the embodiments describedabove can be used to block the interaction of aPC with its substrate,which can include Factor Va or Factor VIIIa.

The monoclonal antibodies have therapeutic use in the treatment ofdisorders of hemostasis such as thrombocytopenia, platelet disorders andbleeding disorders (e.g., hemophilia A, hemophilia B and hemophilia C).Such disorders can be treated by administering a therapeuticallyeffective amount of the anti-aPC monoclonal antibody to a patient inneed thereof. The monoclonal antibodies also have therapeutic use in thetreatment of uncontrolled bleeds in indications such as trauma andhemorrhagic stroke. Thus, also provided is a method for shortening thebleeding time comprising administering a therapeutically effectiveamount of an anti-aPC monoclonal antibody to a patient in need thereof.

In another embodiment, the anti-aPC antibody can be useful as anantidote for aPC-treated patients, including for example wherein aPC isused for the treatment of sepsis or bleeding disorder.

The antibodies can be used as monotherapy or in combination with othertherapies to address a hemostatic disorder. For example,co-administration of one or more antibodies with a clotting factor suchas factor VIIa, factor VIII or factor IX is believed useful for treatinghemophilia. In one embodiment, provided is a method for treating geneticand acquired deficiencies or defects in coagulation comprisingadministering (a) a first amount of a monoclonal antibody that binds tohuman tissue factor pathway inhibitor and (b) a second amount of factorVIII or factor IX, wherein said first and second amounts together areeffective for treating said deficiencies or defects. In anotherembodiment, provided is a method for treating genetic and acquireddeficiencies or defects in coagulation comprising administering (a) afirst amount of a monoclonal antibody that binds to human tissue factorpathway inhibitor and (b) a second amount of factor VIII or factor IX,wherein said first and second amounts together are effective fortreating said deficiencies or defects, and further wherein factor VII isnot coadministered. Also included is a pharmaceutical compositioncomprising a therapeutically effective amount of the combination of amonoclonal antibody and factor VIII or factor IX, wherein thecomposition does not contain factor VII. “Factor VII” includes factorVII and factor VIIa. These combination therapies are likely to reducethe necessary infusion frequency of the clotting factor. Byco-administration or combination therapy is meant administration of thetwo therapeutic drugs each formulated separately or formulated togetherin one composition, and, when formulated separately, administered eitherat approximately the same time or at different times, but over the sametherapeutic period.

In some embodiments, one or more antibodies described herein can be usedin combination to address a hemostatic disorder. For example,co-administration of two or more of the antibodies described herein isbelieved useful for treating hemophilia or other hemostatic disorder.

The pharmaceutical compositions can be parenterally administered tosubjects suffering from hemophilia A or B at a dosage and frequency thatcan vary with the severity of the bleeding episode or, in the case ofprophylactic therapy, can vary with the severity of the patient'sclotting deficiency.

The compositions can be administered to patients in need as a bolus orby continuous infusion. For example, a bolus administration of aninventive antibody present as a Fab fragment can be in an amount of from0.0025 to 100 mg/kg body weight, 0.025 to 0.25 mg/kg, 0.010 to 0.10mg/kg or 0.10-0.50 mg/kg. For continuous infusion, an inventive antibodypresent as an Fab fragment can be administered at 0.001 to 100 mg/kgbody weight/minute, 0.0125 to 1.25 mg/kg/min., 0.010 to 0.75 mg/kg/min.,0.010 to 1.0 mg/kg/min. or 0.10-0.50 mg/kg/min. for a period of 1-24hours, 1-12 hours, 2-12 hours, 6-12 hours, 2-8 hours, or 1-2 hours. Foradministration of an inventive antibody present as a full-lengthantibody (with full constant regions), dosage amounts can be about 1-10mg/kg body weight, 2-8 mg/kg, or 5-6 mg/kg. Such full-length antibodieswould typically be administered by infusion extending for a period ofthirty minutes to three hours. The frequency of the administration woulddepend upon the severity of the condition. Frequency could range fromthree times per week to once every two weeks to six months.

Additionally, the compositions can be administered to patients viasubcutaneous injection. For example, a dose of 10 to 100 mg anti-aPCantibody can be administered to patients via subcutaneous injectionweekly, biweekly or monthly.

As used herein, “therapeutically effective amount” means an amount of ananti-aPC monoclonal antibody or of a combination of such antibody andfactor VIII or factor IX that is needed to effectively increase theclotting time in vivo or otherwise cause a measurable benefit in vivo toa patient in need. The precise amount will depend upon numerous factors,including, but not limited to the components and physicalcharacteristics of the therapeutic composition, intended patientpopulation, individual patient considerations, and the like, and canreadily be determined by one skilled in the art.

V. Kits

Any of the compositions described herein can be comprised in a kit. Thekits will thus comprise, in suitable container, an antibody and/or anadditional agent. Other components can be included in a kit. Diagnosticand therapeutic kits comprise in suitable container, a pharmaceuticallyacceptable formulation of an antibody in a pharmaceutically acceptableformulation. The kit can have a single container, and/or it can havedistinct container for each compound.

When the components of the kit are provided in one and/or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being one example of a particular embodiment. Theantibody can also be formulated into a syringeable composition, in whichcase, the container can itself be a syringe, pipette, and/or other suchlike apparatus, from which the formulation can be applied to an infectedarea of the body, injected into an animal, and/or even applied to and/ormixed with the other components of the kit.

However, the components of the kit can be provided as dried powder(s).When reagents and/or components are provided as a dry powder, the powdercan be reconstituted by the addition of a suitable solvent. It isenvisioned that the solvent can also be provided in another container.

The container will generally include at least one vial, test tube,flask, bottle, syringe and/or other container, into which theantibody/antibody formulation is placed, suitably allocated. The kitscan also comprise a second container for containing a sterile,pharmaceutically acceptable buffer and/or other diluent.

The kits can also include a means for containing the vials in closeconfinement for commercial sale, such as, e.g., injection and/orblow-molded plastic containers into which the desired vials areretained.

Irrespective of the number and/or type of containers, the kits can alsocomprise, and/or be packaged with, an instrument for assisting with theinjection/administration and/or placement of the ultimate antibodywithin the body of an animal. Such an instrument can be a syringe,pipette, forceps, and/or any such medically approved delivery vehicle.

VI. EXAMPLES

The following examples are included to demonstrate embodiments. Itshould be appreciated by those of skill in the art that the techniquesdisclosed in the examples which follow represent techniques discoveredby the inventor to function well in the practice, and thus can beconsidered to constitute modes for its practice. However, those of skillin the art should, in light of the present disclosure, appreciate thatmany changes can be made in the specific embodiments which are disclosedand still obtain a like or similar result without departing from thespirit and scope.

Example 1—Materials and Methods

Design of Humanized 1573 VH/VL.

Protein and DNA sequences information of mouse aPC monoclonal antibodywere obtained. The humanization design was done using the followingmethod: The VH/VL CDR residues were determined and annotated with Kabatnumbering system (world-wide-web at bioinf.org.uk/abs/#kabatnum). Thecanonical structures of the VH/VL CDRs were determined based on reportsin literature (1-2). Based on VH/VL CDR canonical structures, the humangermline framework acceptors with the same canonical structures wereselected.

1573 sequence was used to blast search PDB database and obtain knownantibody structures sharing the highest sequence identities with thetarget antibody. Based on the output of blast search and sequenceidentity ratio, 1M71, 1M7D, and 1M7I were selected as template for VHmodeling, while 1IQW, 11T9 and 2GCY were selected as template for VLchain modeling. Schrodinger suite software was used to build homologymodels for VL and VH chains, with loop optimization. Then the outputmodels were analyzed with software “contact” in CCP4 suite to give alist of all residues in framework regions that interact with residuesfrom CDR regions within 4 Å. Based on the output of software and visualinspection with the model, the following residues in framework wereidentified as residues that contribute to the supporting of CDR loops.Those were: light chain residues Asp70, Tyr36, Thr69, Phe71, Ile2 andTyr49; heavy chain residues Arg94, Arg38, Glu46, Trp47, Asp73, Arg71,and Trp102. For the design of humanized VH, residues supporting loopstructures and VH/VL interface were identified (InternationalApplication No. WO2008021156). Residues important for loop conformationand VH/VL interface were to be back-mutated. Then the VH sequences withthe back-mutations were aligned with the selected germline sub-family.The identities and similarities to each individual human germlineframework sequences within the same canonical subsets were analyzed andthe germline sequence with the best overall homology to the murine VHsequence was identified. It was selected as the acceptor human germlineframework for grafting VH CDRs. Additional considerations for mutationsincluded a Q1E mutation used to eliminate N-terminal pyroglutamateformation. Mutations also included those to maintain consensus withinthe selected VH family, for CDR canonical structures and VH/VLinterface. Mutations might also include those identified as within 4 Åfrom the CDR binding region according to molecular modeling. Analysiswas performed to make sure no N-linked glycosylation pattern (N-{P}-S/T)was found in the proposed humanized construct.

The human JH region was selected based on best sequence homology. Thehumanized VL sequences were also designed based on such method statedabove.

Generation of HC and LC Expression Plasmids.

Humanized V-region sequences were built using gene de novo synthesisapproach. The PCR amplified VHs were cloned into pCP-hCgl expressionvector by homologous recombination. Amplified Vks were cloned intopCP-hck vector using same method.

The variable regions of chimera HC and LC (VH and VL) were PCR-amplifiedfrom 1573. Gel-purified PCR product was cloned into the same vectors ashumanized V-region by homologous recombination.

Transient Transfection of HEK293 Cells.

Approximately 24 hrs before transfection, pass FreeStyle™ 293E at0.5×10⁶ cells/ml and cells were sharked at 120 rpm/min, at 37° C., 8%CO₂. On the day of transfection, the cell density should be about1.0-1.2×10⁶/ml. The cells were split to 1×10⁶/ml with growth medium. Toensure optimal transfection, viability of the cells was determined tobe >95%. DNA was diluted in FreeStyle™ 293 expression medium (293E) in avolume equivalent to one-tenth of culture transfected. PEI was addedinto DNA; the mixture was vortex immediately and incubated for 10 min atroom temperature prior to its addition to the cells. The finalconcentration of DNA to PEI ratio was 1:2.

Purification of Humanized 1573 IgG Antibodies.

Conditioned medium above on day 6 was loaded onto a 1 ml Protein Acolumn, which was pre-equilibrated by 10 ml PBS, pH7.0. The column wasthen washed with equilibrating buffer to baseline after sample loading.After washed, the column was eluted with 100 mM Glycin-HCl pH3.0,followed with immediate addition of 1M Tris-HCl solution to adjust pHvalue to 8.0. The final product was dialyzed against PBS solution.Protein purity was analyzed by SDS-PAGE, SEC and its concentration wasdetermined by Bradford method.

Size Exclusion Chromatography Analysis of the Purified Antibodies.

SEC for analyzing purified antibody was carried out with a Superdex 2005/150, GL column using a HPLC system (LC-20AD, Shimadzu) at ambienttemperature. PBS buffer pH 7.0, at a flow rate 0.3 mL/min was used asthe mobile phase. The protein detection was under 280 nm.

Immobilization of Anti-Mouse FC Antibody onto CM5 Chip.

A CM5 sensor chip was activated in FC2 by 6-min injection (10 μl/min) offreshly prepared 1:1 50 mM NHS: 200 mM EDC. Then anti mouse FC antibodyin 10 mM sodium acetate buffer PH 5.0 (1.4 μl diluted in 90 μl NaAc, pH5.0) was injected onto the activated chip at 5 μl/min (HBS-EP runningbuffer: 10 mM HEPES, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% surfactantP20). The remaining active coupling sites were blocked with 7 mininjection of 1M ethanolamine at 10 μl/min. About 9200 RU was produced.

Immobilization of Anti-Human FC Antibody onto CM5 Chip.

A CM5 sensor chip was activated in FC2 by 7-min injection (10 μl/min) offreshly prepared 1:1 50 mM NHS: 200 mM EDC. Then anti human FC antibodyin 10 mM sodium acetate buffer PH 5.0 (2.5 μl diluted in 90 μl NaAc, pH5.0) was injected onto the activated chip at 5 μl/min (HBS-EP runningbuffer: 10 mM HEPES, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% surfactantP20). The remaining active coupling sites were blocked with 7 mininjection of 1M ethanolamine at 10 μl/min. About 6400 RU was produced.

Biacore Analysis of Human aPC Binding to 1573 Antibodies.

1573 antibody was first captured on the anti-human FC IgG coated CM5chip, followed by injection of antigen human aPC at concentration of 0,1.25, 2.5, 5, 10 and 20 nM. Cycle conditions were as follows: 30 μl/minfor 180 s of association phase and 500 s of dissociation phase. Thesurface was regenerated with a 30 s injection of Gly pH1.5 at 10 μl/min.HBS-EP running buffer: 10 mM HEPES, pH 7.4, 150 mM NaCl, 3.4 mM EDTA,0.005% surfactant P20. Kinetics was calculated with Biacore X100evaluation software ver2.0.

Biacore Analysis of Cyno aPC Binding to 1573 Antibodies.

1573 antibody was first captured on the anti-human FC IgG-coated CM5chip, followed by injection of antigen cyno aPC at concentration of 0,5, 10, 20, 40, 80 nM. Cycle conditions were as follows: 30 μl/min for180 s of association phase and 420 s of dissociation phase. The surfacewas regenerated with a 45 s injection of Gly pH1.5 at 10 μl/min.Kinetics was calculated with Biacore X100 evaluation software ver2.0.

Binding ELISA of Purified Antibodies.

Plates (Nunc, cat#442404) were coated with 100 μl of human aPC (1ng/ml), hPC (2 ng/ml), monkey aPC (2 ng/ml), or monkey PC (2 ng/ml)diluted in DPBS (Gibco, cat#14040) overnight (o/n) at 4° C. Afterwashing, the ELISA plate was blocked with 200 μl MPBST for 1 hr at RT,and tapped dry on a stack of paper towels. To each well 100 μl of IgG tobe tested was added, and incubated 1 hr at RT (for EC₅₀ determinationstart at 20 nM and do 1:3 dilutions).

Plates were washed 5× with PBST. 100 μl of anti-hIgG Fc-HRP (Sigma,cat#A0170) diluted 1:10000 in PBST was added to each well. Plates werewashed and 100 μl/well of TMB substrate was added and incubated at roomtemperature for 5 min. 100 μl/well of 1N HCl was added to terminatereaction. The plate was read with an ELISA plate reader (Biotek, Elx405)at 450 nm wavelength.

Example 2—Results

Design and Sequence Analysis of Chimera and Humanized 1573 Antibody.

Variable region sequence annotations with Kabat numbering(world-wide-web at bioinf.org.uk/abs/#kabatnum) CDR residues areunderlined:

VH: (SEQ ID NO: 31; DNA is SEQ ID NO: 33)EVKLEESGGGLVQPGGSMKLSCVASGFTFSNYYLNWVRQSPEKGLEWVADIRLKSNNYEKHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCIR EGDYFDYWGQGTTLTVSSVL: (SEQ ID NO: 32; DNA is SEQ ID NO: 34)NIVLTQSPASLAVSLGQRATISCRASESVDSFGATFMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNNEDPY TFGGGTKLEIKR1573 VH canonical structure: 1-4-based on H1 and H2 canonicalstructures, the VH can be humanized to subset of the VH3 germlineframework sequences. 1573 Vk canonical structure: 4-1-1-based on a 4-1-1Vk CDR canonical structure, the Vk can be humanized to subset of theVKII germline framework sequences.

1573 VH Humanization Design.

Germline VH3-72 had the best overall homology with the 1573 VH sequence.It was selected as the acceptor human germline framework for grafting1573 VH CDR sequences. hJH6 will be used for 1573 humanization due tobest homology. All possible back-mutations are listed below.

-   -   G49A—Vernier Zone residue, canonical residue (effect CDR H2,        score 3), human residue.    -   N76S—Canonical residue (effect CDRH1, score 3), human residue    -   L78V—Vernier Zone residue, canonical residue (effect CDRH1,        score 3), human residue    -   A93I—Vernier zone residue. Canonical residue (effect CDR H3,        score 3), human residue.        Humanized 1573 VH design with CDRs underlined and back-mutations        double-underlined:

1573 VH (SEQ ID NO: 15)EVKLEESGGGLVQPGGSMKLSCVASGFTFSNYYLNWVRQSPEKGLEWVADIRLKSNNYEKHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCIR EGDYFDYWGQGTTLTVSSH1573 VH.1 (SEQ ID NO: 16)EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYLNWVRQAPGKGLEWVGDIRLKSNNYEKHYAESVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAR EGDYFDYWGQGTTVTVSSH1573 VH.1A (SEQ ID NO: 17)EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYLNWVRQAPGKGLEWVGDIRLKSNNYEKHYAESVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYC

R EGDYFDYWGQGTTVTVSS H1573 VH.1B (SEQ ID NO: 18)EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYLNWVRQAPGKGLEWVGDIRLKSNNYEKHYAESVKGRFTISRDDSKNS

YLQMNSLKTEDTAVYYC

R EGDYFDYWGQGTTVTVSS H1573 VH.1C (SEQ ID NO: 19)EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYLNWVRQAPGKGLEWV

D IRLKSNNYEKHYAESVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYC

R EGDYFDYWGQGTTVTVSS H1573 VH.1D (SEQ ID NO: 20)EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYLNWVRQAPGKGLEWVGDIRLKSNNYEKHYAESVKGRFTISRDDSK

SLYLQMNSLKTEDTAVYYC

R EGDYFDYWGQGTTVTVSS H1573 VH.1E (SEQ ID NO: 21)EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYLNWVRQAPGKGLEWVA DIRLKSNNYEKHYAESVKGRFTISRDDSKNS

YLQMNSLKTEDTAVYYC

R EGDYFDYWGQGTTVTVSS H1573 VH.1F (SEQ ID NO: 22)EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYLNWVRQAPGKGLEWVA DIRLKSNNYEKHYAESVKGRFTISRDDSK

S

YLQMNSLKTEDTAVYYC

R EGDYFDYWGQGTTVTVSS VH3-72 (SEQ ID NO: 23)EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMDWVRQAPGKGLEWVGRTRNKANSYTTEYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAR VH3-72/JH6(SEQ ID NO: 24) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMDWVRQAPGKGLEWVGRTRNKANSYTTEYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAR -------WGQGTTVTVSS

1573 Vk Humanization Design.

The Vk germline A2 had the best overall homology to the 1573 Vksequence. It was selected as the acceptor human germline framework forgrafting 1573 Vk CDR sequences. hJk2 will be used for 1573 humanizationdue to best homology. Residues important for loop conformation and VH/VLinterface are highlighted in yellow and the CDRs with the Kabat numbersin red.

Possible back-mutations in 1573Vk humanization design:

M4L—Vernier zone residue (effect CDRL1, 3, score 3), human residue

G68R—Vernier zone residue

Humanized 1573 Vk design with CDR's underlined and back-mutations indouble-underline:

1573 Vk (SEQ ID NO: 25) NIVLTQSPASLAVSLGQRATISCRASESVDSFGATFMH-WYQQKPGQPPKLLIYLASNLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNNEDPYTFGGGTKLEIKR H1573Vk.2 (SEQ ID NO: 26)DIVMTQTPLSLSVTPGQPASISCRASESVDSFGATFMH-WYLQKPGQPPQLLIYLASNLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQNNEDPYTFGQGTKLEIKR H1573Vk.2A (SEQ ID NO: 27) DIV

TQTPLSLSVTPGQPASISCRASESVDSFGATFMH-WYLQKPGQPPQLLIYLASNLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQNNEDPYTFGQGTKLEIKR H1573Vk.2B (SEQ ID NO: 28)DIVMTQTPLSLSVTPGQPASISCRASESVDSFGATFMH-WYLQKPGQPPQLLIYLASNLESGVPDRFSGSGS

TDFTLKISRVEAEDVG VYYCQQNNEDPYTFGQGTKLEIKR H1573Vk.2C (SEQ ID NO: 29) DIV

TQTPLSLSVTPGQPASISCRASESVDSFGATFMH- WYLQKPGQPPQLLIYLASNLESGVPDRFSGSGS

TDFTLKISRVEAEDVG VYYCQQNNEDPYTFGQGTKLEIKR A2 (SEQ ID NO: 30)DIVMTQTPLSLSVTPGQPASISCKSSQSLLHSDGKTYLYWYLQKPGQPPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQSIQLPNo N-linked glycosylation pattern (N-{P}-S/T) was found in the proposedhumanized construct in both VH and VL.

Humanized VH and VL Cloning.

All humanized VH and VL were built by gene de novo synthesis approachand clone them into 293 expression vector pCp. Constructs for humanizedantibody are summarized in Table 2:

TABLE 2 Construct summary for 1573 humanization VH (VH3-72) +Construction VL (A2) + Construction JH6/FW4 status JK2/FW4 statusH1573VH.1 ✓ H1573Vk.2 ✓ H1573VH.1A ✓ H1573Vk.2A ✓ H1573VH.1B ✓H1573Vk.2B ✓ H1573VH.1C ✓ H1573Vk.2C ✓ H1573VH.1D ✓ 1573Vk (chimeric) ✓H1573VH.1E ✓ H1573VH.1F ✓ 1573VH (chimeric) ✓

Expression and Purification of Chimera and Humanized 1573 Antibody.

293 transfection designs were summarized in Table 3. Conditioned medium(Method 3.3) on day 6 was loaded onto a 1 ml Protein A column.Recombinant 1573 antibody was collected, purified. The chimera andhumanized 1573 IgG proteins all showed good expression levels.

TABLE 3 HEK293 co-transfection design VH 1573VH VK H1573VH.1 H1573VH.1AH1573VH.1B H1573VH.1C H1573VH.1D H1573VH.1E H1573VH.1F chimericH1573Vk.2 Hu1573-3 Hu1573-6 Hu1573-9 Hu1573-12 Hu1573-15 H1573Vk.2AHu1573-4 Hu1573-7 Hu1573-10 Hu1573-13 Hu1573-16 H1573Vk.2B Hu1573-5Hu1573-8 Hu1573-11 Hu1573-14 Hu1573-17 H1573Vk.2C Hu1573-1 Hu1573-21573Vk chimetric

SEC Characterization of Purified Chimera and Humanized 1573 Antibody.

To evaluate the purity and percentage of aggregation of the purifiedchimera 1573 and humanized antibodies, the samples were loaded onto SECrespectively (FIG. 1). All CP generated antibodies was eluted as asingle sharp peak around 5.75 min with PBS pH 7.0 buffer. The elutiontime is very close to that of one of the protein markers (158 kDa,elution time is 5.85 min), suggesting most of them show more than 90% ofmonomer, two antibodies show more than 80% monomer.

Biacore Analysis of 1573 Binding to the Humanized Antibodies.

aPC was immobilized directly onto a new CM5 chip using amine couplingmethod. After overnight equilibrium, 100 nM 1573 antibodies wereinjected over the surface and strong signal was detected. Therefore, theaffinity determination was done using this chip. Each of antibody wasinjected over the chip surfaces respectively at 30 μl/min for 180 s ofassociation phase and 300 s of dissociation phase.

TABLE 4 Kinetic parameters for interaction between 1573 antibodies andantigen hAPC cyno APC determined by surface plasmon resonance (data fitusing 1:1 binding model) Binding to human APC Binding to cyno APCOverall Overall Antibody affinity affinity Code ka (1/Ms) kd (1/s) KD(M) ka (1/Ms) kd (1/s) KD (M) 1573 Chimeric 2.32E+06 0.008112 3.49E−095.55E+05 0.007664 1.38E−08 1573 mouse 9.48E+05 0.005199 5.49E−093.44E+05 0.007254 2.11E−08 Hu 1573-1 1.46E+06 0.005538 3.80E−09 2.40E+050.004016 1.67E−08 Hu 1573-2 1.35E+06 0.00624 4.61E−09 2.35E+05 0.0038431.64E−08 Hu 1573-3 1.44E+06 0.008182 5.70E−09 7.43E+05 0.01053 1.42E−08Hu 1573-4 1.52E+06 0.007723 5.10E−09 7.43E+05 0.01053 1.42E−08 Hu 1573-51.54E+06 0.006397 4.16E−09 2.02E+05 0.002926 1.45E−08 Hu 1573-6 1.43E+060.008477 5.94E−09 6.27E+05 0.007074 1.13E−08 Hu 1573-7 1.36E+06 0.0085746.33E−09 2.55E+05 0.004707 1.85E−08 Hu 1573-8 1.49E+06 0.005995 4.03E−093.63E+05 0.004466 1.23E−08 Hu 1573-9 1.45E+06 0.008152 5.63E−09 4.75E+050.007239 1.52E−08 Hu 1573-10 1.45E+06 0.006364 4.38E−09 4.34E+050.004786 1.10E−08 Hu 1573-11 1.38E+06 0.008958 6.49E−09 2.69E+050.004199 1.56E−08 Hu 1573-12 1.40E+06 0.008443 6.03E−09 3.37E+050.005404 1.61E−08 Hu 1573-13 1.40E+06 0.006553 4.69E−09 2.29E+05 0.003431.50E−08 Hu 1573-14 1.40E+06 0.008559 6.13E−09 2.99E+05 0.0052411.76E−08 Hu 1573-15 1.37E+06 0.008448 6.15E−09 2.25E+05 0.0045242.02E−08 Hu 1573-16 1.42E+06 0.006434 4.52E−09 2.36E+05 0.0037251.58E−08 Hu 1573-17 1.45E+06 0.008329 5.75E−09 2.69E+05 0.0041991.56E−08

Binding ELISA of Purified Antibodies.

For human PC and monkey PC as negative controls, humanized antibodies atdifferent concentrations showed very weak response. They have no bindingto the human PC and monkey PC antigen. The results were showed in FIG. 5and FIG. 6. For human aPC and monkey aPC, all the humanized antibodiesshowed good binding affinity, it confirmed the Biacore analysis (Table4). With the dilution curve of humanized antibodies, the inventors alsocalculated the EC₅₀ of each antibody, which are shown in Table 5.

Chimera and humanized 1573 are cloned in CP's expression vector andgenerated in CP. The interaction between aPC and antibodies includingchimera and humanized are characterized as fast association and fastdissociation by Biacore assay. Furthermore, all the humanized 1573variants fulfilled affinity criteria. Hu1573-1, 2, 5, 8, 10 could begood candidates, as they have affinity higher than 5 nM and with only2-3 back-mutations.

TABLE 5 EC₅₀ of 1573 humanized antibodies Cyno-APC Cyno-PC Human-APCHuman-PC Humanized Ab EC50 (nM) EC50 (nM) EC50 (nM) EC50 (nM) 1573chimeric 0.8302 No binding 0.3337 No binding Hu1573-1 0.8647 No binding0.3693 No binding Hu1573-2 0.7745 No binding 0.2652 No binding Hu1573-30.8723 No binding 0.5915 No binding Hu1573-4 0.9041 No binding 0.5834 Nobinding Hu1573-5 1.0350 No binding 0.5520 No binding Hu1573-6 0.8996 Nobinding 0.2741 No binding Hu1573-7 0.8525 No binding 0.4446 No bindingHu1573-8 0.7129 No binding 0.6972 No binding Hu1573-9 0.9857 No binding0.3134 No binding Hu1573-10 0.6800 No binding 0.5752 No bindingHu1573-11 1.466 No binding 0.6868 No binding Hu1573-12 0.6418 No binding0.3394 No binding Hu1573-13 0.8045 No binding 0.2356 No bindingHu1573-14 0.9838 No binding 0.2247 No binding Hu1573-15 0.8891 Nobinding 0.2880 No binding Hu1573-16 1.2290 No binding 0.2328 No bindingHu1573-17 1.2100 No binding 0.6267 No binding

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods have beendescribed in terms of specific embodiments, it will be apparent to thoseof skill in the art that variations can be applied to the compositionsand/or methods and in the steps or in the sequence of steps of themethod described herein without departing from the concept, spirit andscope of the disclosure. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related canbe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept as defined by the appended claims.

The invention claimed is:
 1. A humanized antibody binding to activatedprotein C comprising: (a) a heavy light chain comprising heavy lightchain CDRs represented by SEQ ID NOS: 1, 2 and 3; and wherein the lightchain framework regions are represented by amino acid residues 1 to 23,39 to 53, 61 to 92 and 102 to 112 of SEQ ID NO: 26, or having 5 or fewerconservative amino acid substitutions to SEQ ID NO: 26 frameworkregions, and (b) a light heavy chain comprising light heavy chain CDRsrepresented by SEQ ID NOS: 4, 5 and 6 and wherein the heavy chainframework regions are represented by amino acid residues 1 to 30, 36 to49, 69 to 100 and 108 to 118 of SEQ ID NO: 16, or having 5 or fewerconservative amino acid substitutions to SEQ ID NO: 16 frameworkregions.
 2. The antibody of claim 1, wherein the heavy chain frameworkregions are represented by SEQ ID NOS: 7, 8, 9 and 10, or having 5 orfewer conservative amino acid substitutions.
 3. The antibody of claim 1,wherein the light chain framework regions are represented by SEQ ID NOS:11, 12, 13 and 14, or having 5 or fewer conservative amino acidsubstitutions.
 4. The antibody of claim 2 1, wherein residue 14 49 ofSEQ ID NO: 8 16 is substituted with Alanine.
 5. The antibody of claim 21, wherein residues 11, 13 and 31 of SEQ ID NO: 9 79, 81 and 99 of SEQID NO: 16 are is substituted with one or more of Serine (residue 11 79),Valine (residue 13 81) and Isoleucine (residue 31 99).
 6. The antibodyof claim 1, wherein said heavy chain comprises SEQ ID NOS: 16-24 NO: 16,17, 18, 19, 20, 21 or
 22. 7. The antibody of claim 3 1, wherein residue4 of SEQ ID NO: 11 26 is substituted with Leucine.
 8. The antibody ofclaim 3 1, wherein residue 12 72 of SEQ ID NO: 13 26 is substituted withArginine.
 9. The antibody of claim 1, wherein said light chain comprisesSEQ ID NOS: 26-30 NO: 26, 27, 28 or
 29. 10. The antibody of claim 1,wherein the antibody is a single-chain antibody or antibody fragmentbinding activated protein C.
 11. The antibody of claim 10, wherein theantibody fragment is further defined as Fab′, Fab, F(ab′)₂, a singledomain antibody, Fv, or scFv.
 12. A cell or cell line comprising anucleic acid encoding a humanized antibody binding to activated proteinC comprising: (a) a heavy light chain comprising heavy light chain CDRsrepresented by SEQ ID NOS: 1, 2 and 3; and wherein the light chainframework regions are represented by amino acid residues 1 to 23, 39 to53, 61 to 92 and 102 to 112 of SEQ ID NO: 26, or having 5 or fewerconservative amino acid substitutions to SEQ ID NO: 26 frameworkregions, and (b) a light heavy chain comprising light heavy chain CDRsrepresented by SEQ ID NOS: 4, 5 and 6, wherein the heavy chain frameworkregions are represented by amino acid residues 1 to 30, 36 to 49, 69 to100 and 108 to 118 of SEQ ID NO: 16, or having 5 or fewer conservativeamino acid substitutions to SEQ ID NO: 16 framework regions.
 13. Theantibody of claim 1, dispersed in a pharmaceutically acceptable carrier.14. A method of inhibiting activated protein C anticoagulant activityand/or amidolytic activity in a subject, comprising administering aneffective amount of an antibody according to claim 1 to said subject.15. A method of treating a subject in need of blood coagulationcomprising administering an effective amount of an antibody according toclaim 1 to said subject.
 16. The method of claim 15, wherein saidsubject is suffering from hemophilia.
 17. A method of promotinghemostasis or thrombosis in a subject, comprising administratingadministering an effective amount of an antibody according to claim 1 tosaid subject.
 18. The method of claim 17, wherein the subject is atrauma patient.
 19. A kit comprising an antibody according to claim 1.